Protein subunit interactions and structural integrity of amyloidogenic transthyretins: evidence from electrospray mass spectrometry.

Wild-type and variant transthyretins form amyloid fibrils in two different diseases. The biologically active form of transthyretin is a tetramer but there is evidence that a monomeric species is the amyloidogenic intermediate. Using mass spectrometry we have developed an approach to monitor the proportions of monomer and tetramer in wild-type and variant transthyretins, and found a strong correlation between the instability of the tetramer in the gas phase and the amyloidogenicity of the protein variant. The presence of water molecules in the central channel has been found to be critical for maintaining intact the complex in the gas phase, with additional stability observed in the presence of excess thyroxine. The solution structure of monomeric transthyretin under fibril-forming conditions was studied using hydrogen exchange monitored by mass spectrometry. The results show that Val30Met transthyretin, the commonest amyloidogenic variant, exhibits loss of hydrogen exchange protection substantially more rapidly than the wild-type protein, suggesting partial unfolding of the beta-sheet structure. These results provide new insights into the correlation between tetramer stability and amyloidogenicity as well as supporting a possible route to fibril formation via transient unfolding of the transthyretin monomer.